Pharmacological glucocorticoids (GCs) have been effectively used to treat inflammation for over a half century, resulting in the established paradigm of GC action that addresses only mechanisms that lead to suppression of inflammation and immunity. In contrast, and less appreciated, is the fact that physiological stress-associated concentrations of GCs-but not higher pharmacological levels-play an essential enhancing role in the activation of immune and inflammatory responses. We therefore propose a pilot human study to obtain data that will support a new paradigm, one addressing the mechanisms by which a transient in vivo increase in cortisol to stress-associated levels (e.g., levels that follow trauma) prepares for an enhanced innate immune response when a subsequent activation event occurs (e.g., an infection). Our study is designed to test the hypothesis that preparative phenotypic and molecular changes induced by physiological concentrations of cortisol in human monocytes and macrophages, lead to functionally opposing consequences compared with pharmacological GC doses. Our secondary aim, identifying mechanisms for individual-specific differential responses to 'stress- cortisol' versus 'pharm-cortisol' would, if successful, provide a screening approach with far reaching benefits for nearly all avenues of biomedical, behavioral, and clinical research. Specifically, we propose to: 1. Identify specific cellular and molecular changes that distinguish stress-cortisol enhancement from pharm- cortisol suppression of human monocyte/macrophage innate immune activation pathways. We have found that stress-cortisol pretreatment reproducibly enhances LPS-induced IL-6 production by human monocyte-derived macrophages from some subjects (responders) but not others (non-responders). Inter-individual differences and differential effects of stress- vs. pharm-cortisol will be used to discriminate by flow cytometry, gene profiling analysis and ELISA assays the most relevant molecular events that lead to stress-cortisol enhanced cytokine production. We will specifically interrogate mechanisms by which stress-cortisol enhancement of LPS- induced MAP kinase phosphorylation augments innate immune activation in human macrophages. 2. Identify, at the molecular level, specific monocyte responses that are differentially regulated by stress- cortisol and pharm-cortisol in vivo. Each subject will serve as his/her own control, and will receive 3 in vivo treatments; saline, stress-cortisol (40mg/70kg/6hours), or pharm-cortisol (400mg/70kg/6hours) with an inter- treatment interval that returns measurements to baseline. Flow cytometry, Taqman real time PCR and ELISA assays will be used to determine the in vivo onset, magnitude and durability of stress-cortisol versus pharm- cortisol-induced changes in key cellular regulators of innate immune inflammation. We plan to identify specific molecular events that predict differential in vivo effects of stress-cortisol versus pharm-cortisol, or differences in innate immune activation among individual subjects. Our results will direct future studies to determine important clinical distinctions between anti-inflammatory and pro-inflammatory regulatory effects of GCs. PUBLIC HEALTH RELEVANCE: Steroids related to the natural adrenal hormone hydrocortisone have been used to effectively treat inflammation and autoimmunity for over a half century, but unexplained differences in their effectiveness in different people presents a vexing clinical challenge. Improved understanding of the mechanisms by which these steroids regulate mediators of immunity and inflammation is needed to foster new therapeutic interventions for the treatment of acute systemic inflammation. The studies proposed are novel in that, to the best of our knowledge, we will be the first to examine mechanisms by which physiological stress-associated levels of hydrocortisone enhance immune and inflammatory pathways within human monocytic white blood cells in vivo.